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1 Department of Physiology and 2 Second Department of Internal Medicine, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of cAMP accumulation on ATP-dependent priming and Ca2+-dependent fusion in Ca2+-regulated exocytosis were examined in antral mucous cells of guinea pigs by using video-enhanced contrast microscopy. The Ca2+-regulated exocytosis activated by 1 µM ACh consisted of two phases, an initial transient phase followed by a sustained phase, which were potentiated by cAMP accumulation. Depletion of ATP by 100 µM dinitrophenol (uncoupler of oxidative phosphorylation) or anoxia induced the sustained phase without the initial transient phase in Ca2+-regulated exocytosis. However, accumulation of cAMP before depletion of ATP induced and potentiated an initial transient phase followed by a sustained phase in Ca2+-regulated exocytosis. This suggests that the initial transient phase of Ca2+-regulated exocytosis is induced by fusion of all primed granules maintained by ATP and that accumulation of cAMP accelerates ATP-dependent priming of the exocytotic cycle. Moreover, ACh and Ca2+ dose-response studies showed that accumulation of cAMP shifted the dose-response curves to the low concentration side, suggesting that it increases Ca2+ sensitivity in the fusion of the exocytotic cycle. In conclusion, cAMP accumulation increases the number of primed granules and Ca2+ sensitivity of the fusion, which potentiates Ca2+-regulated exocytosis in antral mucous cells.
acetylcholine; adenosine 3',5'-cyclic monophosphate; gastric mucin
secretion; 
-adrenergic agonist; ATP; fusion; priming
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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GASTRIC MUCINS, WHICH ARE high-molecular-weight glycoproteins, are released into the lumen by exocytosis. Exocytosis is reported to be regulated by exocytosis-related proteins, activities of which are regulated by intracellular Ca2+ concentration ([Ca2+]i), protein kinase A (PKA), protein kinase C, G proteins, and ATP (14, 16, 20). In antral mucous cells, mucin secretion has been reported to be maintained by two basic mechanisms, Ca2+-regulated exocytosis and cAMP-regulated exocytosis (5, 12, 13). Our previous reports showed that ACh activated Ca2+-regulated exocytosis mediated via the cholinergic receptor (5, 12) and that PGE2 activated cAMP-regulated exocytosis mediated via the prostanoid EP4 receptor (13). With maximum stimulation, the frequency of cAMP-regulated exocytosis was 5-10% of Ca2+-regulated exocytosis. Thus Ca2+-regulated exocytosis is a major mechanism for mucin release from the antral mucous cells.
The Ca2+-regulated exocytosis in antral mucous cells consisted of two phases: an initial transient increase followed by a sustained increase. The biphasic increase in Ca2+-regulated exocytosis is not induced by a biphasic change in [Ca2+]i, since [Ca2+]i is sustained at a high level during ACh stimulation. Similar observations were reported in parotid and submandibular acinar cells during muscarinic stimulation (11, 22). In pancreatic acinar cells, the granules released their contents via three steps: the first is called docking, the second, priming, and the last, fusion. The priming step is regulated by ATP, and the fusion step is regulated by Ca2+ (14, 16, 20). This indicates that the frequency of exocytosis depends on the number of primed granules, maintaining [Ca2+]i at a high value. In parotid acinar cells, the biphasic responses are suggested to be caused by changes in the number of granules in the prefusion state (4, 22). On the basis of these observations, it seems that a reduction in the intracellular ATP level may alter the biphasic response in Ca2+-regulated exocytosis of antral mucous cells.
Accumulation of cAMP is stimulated by many agonists in gastric mucosa,
such as -adrenergic agonists, PGE2, gastrin,
secretin, and histamine (3, 8, 13). In antral
mucous cells, isoproterenol (IPR, a -adrenergic agonist) potentiated
ACh-evoked exocytotic events, although it did not activate exocytosis.
In salivary acinar cells, IPR was reported to potentiate
Ca2+-regulated exocytosis mediated via cAMP accumulation
(21, 22). This suggests that IPR-stimulated cAMP
accumulation potentiates the frequency of Ca2+-regulated
exocytotic events in antral mucous cells. The question here is why cAMP
accumulation potentiates Ca2+-regulated exocytosis.
Video-enhanced contrast (VEC) optical microscopy enabled us to observe exocytotic events in living cells with high time resolution (1, 5, 11, 18). VEC microscopy is used to observe exocytosis in various exocrine cells, such as salivary acinar cells (10, 11, 15), antral mucous cells (5, 13), frog esophageal pepsinogen-secreting cells (17), and pancreatic acinar cells (2, 6, 7). In the present study, we used VEC microscopy to observe the exocytosis from guinea pig antral mucous cells stimulated with ACh and IPR. The goal of this study is to answer two questions: why Ca2+-regulated exocytosis shows a biphasic response and why IPR potentiates Ca2+-regulated exocytosis in antral mucous cells.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Solutions and chemicals.
Solution I contained (in mM): 121 NaCl, 4.5 KCl, 25 NaHCO3, 1 MgCl2, 1.5 CaCl2, 5 NaHEPES, 5 HHEPES, and 5 glucose. To prepare Ca2+-free
solutions, CaCl2 was removed from solution I and
1 mM EGTA was added. To maintain the Ca2+ concentration of
test solutions at <10 µM, an appropriate amount of
CaCl2, calculated using a computer program, was added into the Ca2+-free solution (9). Solutions were all
adjusted to pH 7.4 by adding HCl (1 M). The
HCO
Cell preparations. Hartley strain male guinea pigs weighing ~250 g were purchased from Shimizu Experimental Animals (Kyoto, Japan) and were kept on standard pellet food and water. Guinea pigs were anesthetized by inhalation of ether, after which they were killed by cervical dislocation. The experiments were approved by the Animal Research Committee of Osaka Medical College, and the animals were cared for according to the guidelines of this committee. The procedures for cell preparations have been previously described in detail (5, 12, 13). Briefly, the antrum was excised and the mucosal layer was stripped from the muscle layer in cooled saline (4°C) by using glass slides. The stripped antral mucosa was minced and then incubated in solution I containing 0.1% collagenase and 2% BSA for 10 min at 37°C. The digested mucosa was then filtered through a nylon mesh with a pore size of 150 µm2 and washed three times. The cells were resuspended in solution I containing 2% BSA (4°C). The suspension was stored at 4°C and used within 3 h in the experiments.
For histological examinations, the stripped mucosa and the isolated cells were fixed in a 150-mM phospate buffered solution containing 10% formaldehyde for 1 day, and they were dehydrated and embedded in paraffin according to the standard protocol. The sections were then stained with hematoxylin-eosin (H-E) or periodic acid-Schiff (PAS) reagent.Observation of exocytosis.
Isolated antral mucous cells were mounted on a coverslip precoated with
neutralized Cell-Tak (Becton Dickinson Labware, Bedford, MA) for firm
attachment of the cells. The coverslip with cells was set in a
perfusion chamber that was mounted on the stage of a differential
interference contrast microscope (BX50Wi; Olympus, Tokyo, Japan)
connected to a VEC system (ARGUS-10; Hamamatsu Photonics, Hamamatsu,
Japan). Images were recorded continuously using a video recorder. The
experiments were performed at 37°C. The volume of the perfusion
chamber was ~20 µl, and the rate of perfusion was 200 µl/min. The
exocytotic events, which were detected by rapid changes in the light
intensity of the granules (5, 10, 11, 15, 18), were
counted in 5-6 cells every 30 s and were normalized to the
cell number (events · cell
1 · 30 s1). The frequencies of exocytotic events in 3-7
experiments were expressed as means ± SE. The peak frequency
observed within 2 min from the start of stimulation was used for the
initial peak frequency, and the frequency observed 4 min after the
start of stimulation was used for the sustained frequency
(events · cell1 · 30 s1).
[Ca2+]i measurements. The isolated antral mucous cells were incubated in solution I containing 2% BSA and 2.5 µM fura 2-AM (Dojindo, Kumamato, Japan) for 25 min at room temperature (22-24°C), and they then were washed three times with solution I containing 2% BSA. Fura 2-loaded cells were resuspended and stored in solution I containing 2% BSA at 4°C and mounted on coverslips precoated with neutralized Cell-Tak, and each coverslip with cells was then set in a perfusion chamber, which was then mounted onto the stage of an inverted microscope (IX70; Olympus) connected to an image analysis system (ARGUS/HiSCA, Hamamatsu Photonics) (5, 12). All experiments were performed at 37°C. The volume of the perfusion chamber was ~80 µl, and the rate of perfusion was 500 µl/min. The fura 2 was excited at 340 nm and 380 nm, and emission was measured at 510 nm. The fluorescence ratios (340/380 nm) were calculated and stored in the image analysis system. In the present study, [Ca2+]i were calculated from the calibration curve, which was obtained from the fluorescence ratio (340/380 nm) of the cell-free Ca2+ calibration solutions containing 10 µM fura 2. Solution III contained (in mM): 130 KCl, 20 NaCl, 2 EGTA, and 10 HEPES, and to prepare the cell-free Ca2+-calibration solutions an appropriate amount of CaCl2, calculated using a computer program, was added into solution III (9). The pH was adjusted to 7.05 by adding KOH (1 M). The dissociation constant of Ca2+ and EGTA used was 214 nM (37°C, pH 7.05) (9). One experiment was performed on five to six coverslips, and the typical responses in [Ca2+]i, which were obtained from three cells on a coverslip, are shown in the figures.
The statistical significance of the difference between the mean values was assessed using Student's t-test. Differences were considered significant at P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present experiments, the concentration of ACh used was 1 µM and that of IPR was 10 µM.
Histological examinations and video images.
The thin sections of stripped antral mucosa were stained with H-E (Fig.
1A) and PAS (Fig.
1C). The PAS-positive antral mucous cells lined the apical
surface of the mucosa (Fig. 1C). Glandular columns, which
were isolated by collagenase treatment, used for experiments are shown
in Fig. 1B (H-E staining) and Fig. 1D (PAS staining). The antral mucous cells having PAS-positive mucous granules
are positioned from the middle to the top of the isolated glandular
column. Thus antral mucous cells having mucous granules were easily
distinguished from other cells having no granules in the isolated
glandular columns (Fig. 1, B and D).
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IPR potentiation of ACh-evoked exocytosis.
Isolated antral mucous cells were stimulated with 10 µM IPR or 1 µM
ACh. Stimulation with IPR alone did not increase exocytotic events
(Fig. 3A). Stimulation with
ACh induced biphasic increases in the frequencies of exocytotic events:
an initial transient phase followed by a sustained phase. The sustained
phase was maintained for ~10 min during ACh stimulation (Fig.
3B). The frequencies of the initial transient phase and the
sustained phase were 9.3 ± 0.82 and 2.3 ± 0.68 events · cell1 · 30 s1
(n = 5), respectively. The effect of IPR on the
ACh-evoked exocytotic events was examined. The addition of IPR 5 min
before ACh stimulation potentiated the biphasic increases in the
frequency of ACh-evoked exocytotic events (Fig. 3C). The
frequencies of the initial transient phase and the sustained phase were
15.2 ± 1.1 and 5.2 ± 0.78 events · cell1 · 30 s1
(n = 7), respectively. Thus IPR potentiated the
frequencies of ACh-evoked exocytotic events; however, it did not alter
their time course, that is, the biphasic response. The IPR-induced
potentiation was inhibited by pretreatment with 10 µM propranolol (an
inhibitor of the -adrenergic receptor).
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1 · 30 s1),
but it caused a gradual decrease in the sustained phase (from 6 to 3 events · cell1 · 30 s1),
and the frequency of exocytotic events reached a plateau value (1.5-2 events · cell1 · 30 s1) within 6 min. On the basis of this IPR dose-response
study, we used 10 µM IPR in the present study.
Accumulation of cAMP during IPR stimulation.
Stimulation with both IPR and ACh also potentiated the frequencies of
exocytotic events similar to those shown in Fig. 3C, and the
frequencies of the initial transient phase and the sustained phase were
14.7 ± 1.3 and 5.2 ± 0.87 events · cell1 · 30 s1
(n = 4), respectively. Thus the potentiation was also
observed when both ACh and IPR were added simultaneously (Fig.
4A). The effects of H-89 (an
inhibitor of PKA) on the potentiation of ACh-evoked exocytotic events
caused by IPR were examined. Our previous report showed that 20 µM
H-89 completely inhibited PKA actions in antral mucous cells
(EC50 = 2.4 µM) (13). Cells were
perfused with solution I containing 20 µM H-89 for 5 min
and then stimulated with both IPR and ACh. In the presence of 20 µM
H-89, the addition IPR did not potentiate the frequencies of ACh-evoked
exocytotic events (Fig. 4A), and the frequencies of the
initial transient phase and the sustained phase were 7.4 ± 1.2 and 1.9 ± 0.28 events · cell1 · 30 s1 (n = 4), respectively. Thus H-89
inhibited the potentiation of ACh-evoked exocytotic events induced by
IPR (Fig. 4A). However, the frequency of the initial
transient phase (7.4 ± 1.2 events · cell1 · 30 s1) was
low compared with that shown in Fig. 3B (9.3 ± 0.82 events · cell1 · 30 s1).
Cells were also pretreated with 20 µM H-89 for 5 min, then stimulated
with ACh alone (Fig. 4B). Stimulation with ACh evoked a
biphasic increase in the frequency of exocytotic events. The frequencies of the initial transient phase and the sustained phase were
6.1 ± 1.3 and 1.3 ± 0.4 events · cell1 · 30 s1
(n = 5), respectively. Thus 20 µM H-89 significantly
decreased the initial peak frequency of ACh-evoked exocytotic events by 20-30% (P < 0.05), but it did not decrease the
sustained frequency of ACh-evoked exocytotic events (Fig.
4B). Thus H-89 partially inhibited the initial phase of the
ACh-evoked exocytotic events.
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1 · 30 s1) in the presence and absence of extracellular
Ca2+, as previously reported (13), and it
potentiated biphasic increases in the frequencies of exocytotic events
evoked by ACh in a dose-dependent manner (Fig.
5). FK at a concentration >1 µM
potentiated the initial transient phase markedly; however, potentiation
of the sustained phase was small. The small potentiation of the
sustained phase is likely to be caused by a granule depletion induced
by the large initial transient phase. The effects of 0.01 µM FK on
ACh-evoked exocytotic events (Fig. 5C) were similar to those
of IPR (Fig. 3C). In the absence of extracellular
Ca2+, FK also potentiated the initial transient phase of
ACh-evoked exocytotic events; however, the sustained phase was not
induced (Fig. 5). Thus the effects of FK on the sustained phase were
detected only in the presence of extracellular Ca2+.
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Effects of IPR on ACh-evoked exocytotic events.
The effect of the ACh dose on exocytotic events was examined in the
presence and the absence of IPR (Fig. 7).
Stimulation with ACh evoked biphasic increases in the frequencies of
exocytotic events, which depended on ACh concentration. Pretreatment
with IPR potentiated both an initial transient phase and a sustained phase in the frequencies of exocytotic events at each ACh concentration tested (Fig. 7, A-D). An addition of IPR
potentiated the increases in frequencies of ACh-evoked exocytotic
events; however, it did not alter the time course of ACh-evoked
exocytosis, that is, an initial transient phase followed by a sustained
phase. The initial peak (1 min after ACh stimulation) and the sustained
frequencies (4 min after ACh stimulation) of ACh-evoked exocytotic
events were plotted against ACh concentrations (Fig. 7. E
and F). The initial peak and the sustained frequencies
increased dose dependently in the presence and absence of IPR, and IPR
shifted the dose-response curve to the left in both the initial
transient phase and the sustained phase. The EC50 of ACh
dose-response curves in the initial and the sustained phases were 1.2 and 1.8 µM in the presence of IPR, whereas they were 5.1 and 8.4 µM
in the absence of IPR (Fig. 7, E and F). Thus IPR
increased the ACh efficacy to the exocytotic responses.
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Effects of dinitrophenol and anoxia.
Experiments were performed in the presence of 100 µM dinitrophenol
(DNP; an uncoupler of oxidative phosphorylation) to inhibit ATP
formation. Cells were perfused with solution I containing 100 µM DNP for 2 min and then stimulated with ACh, which evoked only
the sustained phase and did not induce any initial transient phase
(Fig. 9A). Effects of IPR on
ACh-evoked exocytotic events were also examined in DNP-treated cells
(Fig. 9C). Cells were perfused with solution I
containing 100 µM DNP for 1 min, and IPR was then added 1 min before
ACh stimulation. ACh also evoked only the sustained phase, which was
potentiated (Fig. 9C). Thus in DNP-treated cells, the
initial transient phase in ACh-evoked exocytotic events was eliminated
with or without IPR added before ACh stimulation. The frequency of the
sustained phase remaining was 1.5 ± 0.3 (n = 5)
and 4.6 ± 0.6 events · cell1 · 30 s1 (n = 5) in the absence and presence of
IPR, respectively.
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1 · 30 s1
(n = 4) in the absence and presence of IPR,
respectively. Thus IPR potentiated the sustained phase that remained
under an anoxic condition. The effect of anoxia showed the same effect
as that obtained by DNP.
Similar experiments were also performed in cells stimulated with 1 µM
FK. Cells were first treated with 100 µM DNP for 1 min, and
then 1 µM FK was added 1 min before ACh stimulation, which induced
only the sustained phase. The frequencies of the sustained phase
remaining were potentiated (Fig.
10A). In turn, cells were first perfused with solution I containing 1 µM FK for 1 min, and 100 µM DNP was then added 2 min before ACh stimulation. ACh
evoked biphasic increases in the frequency of exocytotic events, which was much higher than that evoked by ACh alone (Fig. 10B).
These observations suggest that FK potentiates the initial transient increase of ACh-evoked exocytotic events in the absence of DNP (in the
presence of ATP); however, it does not, in the presence of DNP (in the
absence of ATP). DNP in concentrations of <50 µM, did not inhibit
the initial transient increase in ACh-evoked exocytotic events.
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Effects of IPR on
[Ca2+]i.
Effects of IPR on increases in [Ca2+]i evoked
by ACh were examined. IPR alone did not evoke any increase in
[Ca2+]i. ACh increased
[Ca2+]i rapidly and sustained it (Fig.
11A). The
[Ca2+]i at 5 s, 2 min, and 10 min after
ACh stimulation were 171 ± 8.9, 186 ± 4.2, and 134 ± 7.5 nM (n = 5 experiments), respectively. Addition of
IPR did not potentiate increases in [Ca2+]i
evoked by ACh (Fig. 11B). The
[Ca2+]i at 5 s, 2 min, and 10 min after
ACh stimulation were 155 ± 4.5, 168 ± 4.8, and 136 ± 7.8 nM (n = 6 experiments), respectively. In the
absence of extracellular Ca2+, ACh evoked a transient
increase in [Ca2+]i, but the addition of IPR
did not potentiate the transient increase in
[Ca2+]i evoked by ACh (data not shown).
[Ca2+]i in the initial peak (5 s after ACh
stimulation) were 172 ± 3.6 nM (n = 5 experiments) and 163 ± 8.1 nM (n = 4 experiments) in the absence and the presence of IPR, respectively. The
[Ca2+]i were also measured in cells perfused
with the N2-bubbled solution II (an anoxic
condition). ACh increased [Ca2+]i rapidly and
sustained it. ACh-induced increases in
[Ca2+]i under anoxic conditions were similar
to those observed under oxygenated conditions (Fig. 11C).
The [Ca2+]i at 5 s, 2 min, and 10 min
after ACh stimulation were 146 ± 13.5, 171 ± 4.2, and
143 ± 10.0 nM (n = 6 experiments), respectively.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Potentiation of Ca2+-regulated exocytosis. IPR potentiated the frequencies of ACh-evoked exocytotic events in antral mucous cells of guinea pigs. ACh was reported to increase the frequency of exocytotic events mediated by [Ca2+]i (5, 12). The Ca2+-regulated exocytosis in antral mucous cells had characteristic features in its frequency and time course, that is, high frequencies and a biphasic response: an initial transient phase followed by a sustained phase. The initial phase was evoked by Ca2+ release from intracellular stores and Ca2+ influx, from extracellular fluid and the sustained phase, only by Ca2+ influx from extracellular fluid. The accumulation of cAMP was also reported to increase the frequency of exocytotic events (13). The features of cAMP-regulated exocytosis were, however, different from those of Ca2+-regulated exocytosis, that is, low frequency and only a sustained phase (10-20% of the sustained frequency in ACh-evoked exocytotic events). The present study has shown that stimulation with both IPR and ACh evoked biphasic increases in the frequency of exocytotic events, which were higher than those evoked by ACh alone. The responses evoked by both agonists were those of Ca2+-regulated exocytosis in terms of frequency and time course. On the basis of these observations, we concluded that IPR potentiated Ca2+-regulated exocytosis evoked by ACh in antral mucous cells.
IPR-stimulated cAMP accumulation.
IPR is well known to stimulate cAMP accumulation in many cell types.
IPR, however, did not evoke any increase in the frequency of exocytotic
events, whereas 1 µM FK, 500 µM 8Br-cAMP, and 100 µM IBMX
sustained the frequency of exocytotic events (0.2-0.3 events · cell1 · 30 s1).
Potentiation effects of IPR on ACh-evoked exocytotic events were much
less than those of 1 µM FK, 500 µM 8Br-cAMP, and 100 µM IBMX;
however, 0.01 µM FK mimicked the effects of 10 µM IPR. In measuring
cAMP contents in antral mucosal strips, IPR increased the cAMP content
from 3.8 (n = 3) to 6.7 pmol/mg dry wt
(n = 3) (unpublished observations), whereas 10 µM FK
and 1 mM IBMX were reported to increase the cAMP content to 61.3 and
80.8 pmol/mg dry wt, respectively (13). Moreover, 20 µM
H-89 (a PKA inhibitor) eliminated the potentiation of ACh-evoked
exocytotic events by IPR. These observations indicate that IPR
significantly stimulates cAMP accumulation in antral mucous cells.
Ca2+-dependent fusion and ATP-dependent priming. ACh and Ca2+ dose-response studies showed that the frequencies of ACh-evoked exocytotic events depended on [Ca2+]i. Increases in [Ca2+]i evoked by ionomycin or thapsigargin have been reported to activate exocytotic events in antral mucous cells, and removal of extracellular Ca2+ has been reported to inhibit ACh-evoked exocytosis (5). Similar results were also obtained in the present study. Thus the ACh-evoked exocytosis was regulated by [Ca2+]i.
On the other hand, treatment with 100 µM DNP and anoxia (N2 bubbling) eliminated the initial transient increase in the ACh-evoked exocytotic events, although they did not affect [Ca2+]i increases evoked by ACh. DNP is a well-known uncoupler of oxidative phosphorylation, which inhibits ATP formation in the inner mitochondrial membrane because the proton-motive force across the inner mitochondrial membrane is dissipated. Anoxia also inhibits oxidative phosphorylation. Thus inhibition of ATP formation eliminated the initial transient increase in the ACh-evoked exocytotic events; that is, the initial transient increase in Ca2+-regulated exocytosis depends on ATP. It was demonstrated that Ca2+-regulated exocytosis in the pancreatic acinar cell involves at least two biochemically distinct steps (20). The first step requires ATP, but not Ca2+, and primes the exocytotic machinery (therefore called priming), whereas the second step requires Ca2+, but not ATP, and fuses the granule to the plasma membrane (therefore called fusion) (14, 16). The present study has shown that ACh-evoked exocytosis is under regulation by both Ca2+ and ATP in antral mucous cells, suggesting that similar mechanisms regulate Ca2+-regulated exocytosis in antral mucous cells, ATP-dependent priming, and Ca2+-dependent fusion. According to these observations, depletion of ATP decreases the number of primed granules in antral mucous cells and the granules remain unprimed. Under these conditions, increases in [Ca2+]i are unlikely to trigger fusion of granules, since granules are unprimed. In turn, in the presence of ATP, increases in [Ca2+]i immediately trigger fusion of all of the primed granules, which induces the initial transient increase in exocytotic events. However, treatment with DNP or anoxia did not eliminate the sustained increase in ACh-evoked exocytotic events. Even with inhibition of the oxidative phosphorylation, anaerobic glycolysis supplies ATP, the amount of which is small (2 molecules of ATP per 1 molecule of glucose in glycolysis, whereas 36 molecules of ATP per 1 molecule of glucose in complete oxidation). This ATP produced by glycolysis, which may be activated by ACh stimulation, supplies the primed granules, which fuse to the plasma membrane immediately. This maintains the sustained increase of ACh-evoked exocytotic events, even in the absence of oxidative phosphorylation.cAMP modulation of fusion and priming. Accumulation of cAMP stimulated by IPR increased the Ca2+ sensitivity of exocytosis (2.5-10 times), as shown in the ACh and Ca2+ dose-response studies. However, IPR-stimulated cAMP accumulation did not potentiate increases in [Ca2+]i evoked by ACh. Thus potentiations in the frequency of ACh-evoked exocytotic events were not caused by the potentiation of ACh-evoked increases in [Ca2+]i. Yoshimura et al. (21) reported similar observations in amylase secretion in parotid acinar cells, and they concluded that cAMP accumulation increased Ca2+ sensitivity of Ca2+-dependent fusion (4, 21, 22). These observations suggest that cAMP accumulation modulates the Ca2+-dependent fusion of exocytosis in antral mucous cells by increasing Ca2+ sensitivity.
Large accumulation of cAMP, which was stimulated by FK (1 µM), 8Br-cAMP (500 µM), or IBMX (100 µM), induced marked potentiation of the initial transient increase (ATP dependent) in the ACh-evoked exocytotic events, which caused depletion of granules in antral mucous cells (Figs. 5 and 6), but it did not induce any initial transient increase in the ACh-evoked exocytotic events after depletion of ATP. This suggests that this cAMP accumulation in the presence of ATP causes most of the granules to be primed and that an increase in [Ca2+]i immediately triggers fusion of all of the primed granules at once, which evoked the large initial transient increase in the frequency of exocytotic events, as shown in Figs. 5 and 6. Moreover, FK-stimulated cAMP accumulation before ATP depletion (DNP addition) still potentiates the initial transient phase of ACh-evoked exocytosis, whereas that after ATP depletion did not activate the initial transient phase. This suggests that the granules that are primed by FK-stimulated cAMP accumulation in the presence of ATP still remain in the primed condition after depletion of ATP, which induces a small decrease in the number of primed granules. Therefore, the increases in [Ca2+]i triggered the fusion of all of the primed granules, which also induced a large transient increase in the exocytotic events, as shown in Fig. 10. Thus cAMP accumulation accelerates ATP-dependent priming in antral mucous cells. The present study has shown that mucin secretion from the antral mucous cells was potentiated by even a small accumulation of cAMP. There are many agonists that stimulate cAMP accumulation, particularly in the gastric antrum, such as PGE2, gastrin, secretin, and IPR (3, 13). These agonists alone are unlikely to stimulate mucin secretion; however, they potentiate mucin secretion evoked by Ca2+-mobilizing agonists. In antral mucous cells, the biphasic response in Ca2+-regulated exocytosis is induced by the change in the number of primed granules. The initial transient phase is caused by the immediate fusion of the granules that are already in the primed condition, and the sustained increase is caused by fusion of granules that are in the process of priming. Accumulation of cAMP accelerates the priming step, resulting in an increase in the number of primed granules, and also increases the Ca2+ sensitivity of the fusion step. These modulations by IPR-stimulated cAMP accumulation potentiate the frequency of ACh-evoked exocytotic events in antral mucous cells.| |
ACKNOWLEDGEMENTS |
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We thank Drs. H. Yoshida (Dept. of Physiology) and A. Ohnishi (Second Dept. of Internal Medicine) for their technical support.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Nakahari, Dept. of Physiology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki, 569-8686, Japan (E-mail: takan{at}art.osaka-med.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00300.2001
Received 10 July 2001; accepted in final form 15 January 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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); n = 6].
Cells were perfused with H-89 for 5 min and then stimulated with IPR
and ACh. However, addition of IPR did not potentiate the frequency of
ACh-evoked exocytotic events [H-89 (+); n = 4].
B: effects of H-89 (20 µM) on ACh-evoked exocytotic
events. Cells were perfused with H-89 for 5 min and then stimulated
with ACh. The initial peak frequency of ACh-evoked exocytotic events
significantly decreased in H-89 treated cells (P < 0.05), but the sustained frequency did not.
) or absence (
) of IPR (10 µM) are
shown. Cells were stimulated with IPR for 5 min before stimulation with
10 (A; n = 5), 4 (B;
n = 6), 1 (C; n = 6), or 0.4 µM ACh (D; n = 4). IPR potentiated both
phases evoked by ACh. E: initial peak frequencies of
ACh-evoked exocytotic events were plotted against ACh concentrations
(n = 4-6). IPR shifted the dose-response curve to
the left. F: sustained frequencies of ACh-evoked exocytotic
events (4 min after ACh stimulation) were also plotted against ACh
concentrations (n = 4-6). IPR shifted the
dose-response curve to the left. *Significant difference for
IPR-treated vs. -untreated cells.
